Insulin-like growth factor binding proteins and IGFBP proteases: A dynamic system regulating the ovarian folliculogenesis

International audienceThe aim of the present article is to update our understanding of the expression of the insulin-like growth factor binding proteins (IGFBPs), IGFBP proteases and their implication in the different processes of ovarian folliculogenesis in mammals. In the studied species, IGFs and several small-molecular weight IGFBPs (in particular IGFBP-2 and IGFBP-4) are considered, respectively, as stimulators and inhibitors of follicular growth and maturation. IGFs play a key role in sensitizing ovarian granulosa cells to FSH action during terminal follicular growth. Concentrations of IGFBP-2 and IGFBP-4 in follicular fluid strongly decrease during follicular growth, leading to an increase in IGF bioavailability. Inversely, atresia is characterized by an increase of IGFBP-2 and IGFBP-4 levels, leading to a decrease in IGF bioavailability. Changes in intrafollicular IGFBPs content are due to variations in mRNA expression and/or proteolytic degradation by the pregnancy-associated plasma protein-A (PAPP-A), and likely participates in the selection of dominant follicles. The identification of PAPP-A2, as an IGFBP-3 and -5 protease, and stanniocalcins (STCs) as inhibitors of PAPP-A activity extends the IGF system. Studies on their implication in folliculogenesis in mammals are still in the early stages

Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury

Basic fibroblast growth factor (bFGF) and growth and differentiation factor (GDF)-5 stimulate the healing of medial collateral ligament (MCL) injury. However, the effect of isolated and combined use of bFGF/GDF-5 remains still unclear. We investigated cellular proliferation and migration responding to bFGF/GDF-5 using rabbit MCL fibroblasts. Rabbit MCL injury was treated by bFGF and/or GDF-5 with peptide hydrogels. Gene expression and deposition of collagens in healing tissues were evaluated. bFGF/GDF-5 treatment additively enhanced cell proliferation and migration. bFGF/GDF-5 hydrogels stimulated Col1a1 expression without increasing Col3a1 expression. Combined use of bFGF/GDF-5 stimulated type I collagen deposition and the reorganization of fiber alignment, and induced better morphology of fibroblasts in healing MCLs. Our study indicates that combined use of bFGF/GDF-5 might enhance MCL healing by increasing proliferation and migration of MCL fibroblasts, and by regulating collagen synthesis and connective fiber alignment

Action Mechanism of Inhibin α-Subunit on the Development of Sertoli Cells and First Wave of Spermatogenesis in Mice

Inhibin is an important marker of Sertoli cell (SC) activity in animals with impaired spermatogenesis. However, the precise relationship between inhibin and SC activity is unknown. To investigate this relationship, we partially silenced both the transcription and translation of the gene for the α-subunit of inhibin, Inha, using recombinant pshRNA vectors developed with RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Clontech Laboratories, Mountain View, Calif). We found that Inha silencing suppresses the cell-cycle regulators Cyclin D1 and Cyclin E and up-regulates the cell-cycle inhibitor P21 (as detected by Western blot analysis), thereby increasing the number of SCs in the G1 phase of the cell cycle and decreasing the amount in the S-phase of the cell cycle (as detected by flow cytometry). Inha silencing also suppressed Pdgfa, Igf1, and Kitl mRNA levels and up-regulated Tgfbrs, Inhba, Inhbb, Cyp11a1, Dhh, and Tjp1 mRNA levels (as indicated by real-time polymerase chain reaction [PCR] analysis). These findings indicate that Inha has the potential to influence the availability of the ligand inhibin and its antagonist activin in the SC in an autocrine manner and inhibit the progression of SC from G1 to S. It may also participate in the development of the blood–testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa. Real-time PCR and Western blot analyses of Inha, Inhba, and Inhbb mRNA and Inha levels over time show that Inha plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice

Ovotoxic Effects of Galactose Involve Attenuation of Follicle-Stimulating Hormone Bioactivity and Up-Regulation of Granulosa Cell p53 Expression

Clinical evidence suggests an association between galactosaemia and premature ovarian insufficiency (POI); however, the mechanism still remains unresolved. Experimental galactose toxicity in rats produces an array of ovarian dysfunction including ovarian development with deficient follicular reserve and follicular resistance to gonadotrophins that characterize the basic tenets of human POI. The present investigation explores if galactose toxicity in rats attenuates the bioactivity of gonadotrophins or interferes with their receptor competency, and accelerates the rate of follicular atresia. Pregnant rats were fed isocaloric food-pellets supplemented with or without 35% D-galactose from day-3 of gestation and continuing through weaning of the litters. The 35-day old female litters were autopsied. Serum galactose-binding capacity, galactosyltransferase (GalTase) activity, and bioactivity of FSH and LH together with their receptor competency were assessed. Ovarian follicular atresia was evaluated in situ by TUNEL. The in vitro effects of galactose were studied in isolated whole follicles in respect of generation of reactive oxygen species (ROS) and expression of caspase 3, and in isolated granulosa cells in respect of mitochondrial membrane potential, expression of p53, and apoptosis. The rats prenatally exposed to galactose exhibited significantly decreased serum GalTase activity and greater degree of galactose-incorporation capacity of sera proteins. LH biopotency and LH-FSH receptor competency were comparable between the control and study population, but the latter group showed significantly attenuated FSH bioactivity and increased rate of follicular atresia. In culture, galactose increased follicular generation of ROS and expression of caspase 3. In isolated granulosa cells, galactose disrupted mitochondrial membrane potential, stimulated p53 expression, and induced apoptosis in vitro; however co-treatment with either FSH or estradiol significantly prevented galactose-induced granulosa cell p53 expression. We conclude that the ovotoxic effects of galactose involves attenuation of FSH bioactivity that renders the ovary resistant to gonadotrophins leading to increased granulosa cell expression of p53 and follicular atresia

LGR6 Is a High Affinity Receptor of R-Spondins and Potentially Functions as a Tumor Suppressor

BACKGROUND: LGR6 (leucine-rich repeat containing, G protein-coupled receptor 6) is a member of the rhodopsin-like seven transmembrane domain receptor superfamily with the highest homology to LGR4 and LGR5. LGR6 was found as one of the novel genes mutated in colon cancer through total exon sequencing and its promoter region is hypermethylated in 20-50% of colon cancer cases. In the skin, LGR6 marks a population of stem cells that can give rise to all cell lineages. Recently, we and others demonstrated that LGR4 and LGR5 function as receptors of R-spondins to potentiate Wnt/β-catenin signaling. However, the binding affinity and functional response of LGR6 to R-spondins, and the activity of colon cancer mutants of LGR6 have not been determined. PRINCIPAL FINDINGS: We found that LGR6 also binds and responds to R-spondins 1-3 with high affinity to enhance Wnt/β-catenin signaling through increased LRP6 phosphorylation. Similar to LGR4 and LGR5, LGR6 is not coupled to heterotrimeric G proteins or to β-arrestin following R-spondin stimulation. Functional and expression analysis of three somatic mutations identified in colon cancer samples indicates that one mutant fails to bind and respond to R-spondin (loss-of-function), but the other two have no significant effect on receptor function. Overexpression of wild-type LGR6 in HeLa cells leads to increased cell migration following co-treatment with R-spondin1 and Wnt3a when compared to vector control cells or cells overexpressing the loss-of-function mutant. CONCLUSIONS: LGR6 is a high affinity receptor for R-spondins 1-3 and potentially functions as a tumor suppressor despite its positive effect on Wnt/β-catenin signaling

Transforming Growth Factor β Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function

The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus

Activation of latent human GDF9 by a single residue change (Gly(391)Arg) in the mature domain

Growth differentiation factor 9 (GDF9) controls granulosa cell growth and differentiation during early ovarian folliculogenesis and regulates cumulus cell function and ovulation rate in the later stages of this process. Similar to other TGF-β superfamily ligands, GDF9 is secreted from the oocyte in a noncovalent complex with its prodomain. In this study, we show that prodomain interactions differentially regulate the activity of GDF9 across species, such that murine (m) GDF9 is secreted in an active form, whereas human (h) GDF9 is latent. To understand this distinction, we used site-directed mutagenesis to introduce nonconserved mGDF9 residues into the pro- and mature domains of hGDF9. Activity-based screens of the resultant mutants indicated that a single mature domain residue (Gly³⁹¹) confers latency to hGDF9. Gly³⁹¹ forms part of the type I receptor binding site on hGDF9, and this residue is present in all species except mouse, rat, hamster, galago, and possum, in which it is substituted with an arginine. In an adrenocortical cell luciferase assay, hGDF9 (Gly³⁹¹Arg) had similar activity to mGDF9 (EC₅₀ 55 ng/ml vs. 28 ng/ml, respectively), whereas wild-type hGDF9 was inactive. hGDF9 (Gly³⁹¹Arg) was also a potent stimulator of murine granulosa cell proliferation (EC₅₀ 52 ng/ml). An arginine at position 391 increases the affinity of GDF9 for its signaling receptors, enabling it to be secreted in an active form. This important species difference in the activation status of GDF9 may contribute to the variation observed in follicular development, ovulation rate, and fecundity between mammals.Courtney M. Simpson, Peter G. Stanton, Kelly L. Walton, Karen L. Chan, Lesley J. Ritter, Robert B. Gilchrist, and Craig A. Harriso

LA PROTEOLYSE DE L'INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-4 (IGFBP-4) DANS LES FOLLICULES OVARIENS CHEZ LES MAMMIFERES DOMESTIQUES (IDENTIFICATION DE LA PROTEASE, ETUDE DE SA REGULATION, CONSEQUENCES SUR LA MATURATION FOLLICULAIRE)

LES IGFS (INSULIN-LIKE GROWTH FACTORS) JOUENT UN ROLE CRUCIAL DANS LE DEVELOPPEMENT FOLLICULAIRE EN AMPLIFIANT L'ACTION DES HORMONES GONADOTROPES HYPOPHYSAIRES, FSH ET LH. LEUR ACTION STIMULANTE EST MODULEE PAR LA PRESENCE DE PROTEINES DE LIAISON, LES IGF-BINDING PROTEINS (IGFBPS), DANS LE LIQUIDE FOLLICULAIRE. CHEZ LA BREBIS, LA VACHE, LA TRUIE, LA JUMENT ET LA FEMME, LA CROISSANCE ET L'ATRESIE FOLLICULAIRE SONT ASSOCIEES RESPECTIVEMENT A UNE DIMINUTION ET A UNE AUGMENTATION DES TENEURS EN IGFBP-4 DANS LE LIQUIDE FOLLICULAIRE. CES VARIATIONS SONT ESSENTIELLEMENT DUES A DES VARIATIONS DE L'ACTIVITE PROTEOLYTIQUE CAPABLE DE LA DEGRADER. LES OBJECTIFS DE NOTRE TRAVAIL ONT ETE 1) D'IDENTIFIER LA PROTEASE RESPONSABLE DE LA DEGRADATION PROTEOLYTIQUE DE L'IGFBP-4 DANS LES FOLLICULES PREOVULATOIRES ; 2) DE DETERMINER LES MECANISMES LOCAUX DE REGULATION DE L'ACTIVITE PROTEOLYTIQUE ; 3) DE RECHERCHER LE ROLE BIOLOGIQUE DES FRAGMENTS DE PROTEOLYSE DE L'IGFBP-4 DANS LA MATURATION FOLLICULAIRE. NOUS AVONS MONTRE QUE LA PREGNANCY-ASSOCIATED PLASMA PROTEIN-A (PAPP-A) EST LA METALLOPROTEASE IMPLIQUEE DANS LA DEGRADATION DE L'IGFBP-4 DANS LES FOLLICULES PREOVULATOIRES DE BREBIS, DE VACHE, DE TRUIE ET DE JUMENT. CHEZ LA TRUIE ET LA VACHE, L'EXPRESSION DES ARNMS DE LA PAPP-A DANS LES CELLULES DE GRANULOSA EST ETROITEMENT CORRELEE A CELLE DE L'AROMATASE ET DES RECEPTEURS A LH. ELLE EST MAXIMALE DANS LES FOLLICULES PREOVULATOIRES ET FAIBLE, MAIS NON NULLE, DANS LES FOLLICULES ATRETIQUES ET DANS LES FOLLICULES IMMATURES. AU NIVEAU POST-TRADUCTIONNEL, L'IGF-I POTENTIALISE LA PROTEOLYSE DE L'IGFBP-4 PAR LA PAPP-A. A L'INVERSE, LES IGFBP-2 ET -3 AINSI QUE DES PEPTIDES DE LIAISON A L'HEPARINE DERIVES DU DOMAINE CARBOXY-TERMINAL DE L'IGFBP-3 OU -5 OU DU CTGF (CONNECTIVE TISSUE GROWTH FACTOR), DU HIP (HEPARAN/HEPARIN INTERACTING PEPTIDE) ET DE LA VITRONECTINE INHIBENT LA DEGRADATION DE L'IGFBP-4 PAR LE LIQUIDE FOLLICULAIRE DE FOLLICULES PREOVULATOIRES. CES EFFETS PASSENT PAR UNE REDUCTION DE LA BIODISPONIBILITE INTRAFOLLICULAIRE DES IGFS ET/OU PAR UNE INTERACTION DIRECTE AVEC LA PAPP-A PAR L'INTERMEDIAIRE DU DOMAINE DE LIAISON A L'HEPARINE. ENFIN, IN VITRO, DES RESULTATS PRELIMINAIRES MONTRENT UN EFFET POTENTIALISATEUR DES FRAGMENTS CARBOXY-TERMINAUX DE L'IGFBP-4 SUR LA PRODUCTION DE PROGESTERONE PAR LES CELLULES DE GRANULOSA OVINES CULTIVEES EN PRESENCE D'IGF-I, SUGGERANT QUE LA DEGRADATION PROTEOLYTIQUE DE L'IGFBP-4 ABOUTIT A UNE AMPLIFICATION DE L'ACTION DE L'IGF-I EN PARTICIPANT A L'AUGMENTATION DE SA BIODISPONIBILITE ET EN POTENTIALISANT SON ACTION AU NIVEAU CELLULAIRE.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF